A promising direction in the development of anti-cancer drugs with high specificity and low toxicity is the possibility of inhibiting the functions of synthetically lethal (SL) (synergetic) DNA Damage and Repair (DDR) drug targets that are specifically modulated in cancer development. As outlined in the 290 SBIR contract proposals, one of the promising, although unrealized, strategies for identifying SL targets is to apply a systematic, unbiased RNAi viability screen based on the ablation of two targets from a complete set of DDR genes. As a first step for constructing a cancer-SL gene interaction map, which used Phase I SBIR contract funding, we developed the technology for the construction of novel bi-specific pooled lentiviral shRNA libraries designed for the combinatorial knockdown of any two targets in a set of rationally selected genes. Furthermore, we successfully demonstrated the application of the bi-specific shRNA library to detect SL binary interactions between 40 DDR genes in an isogenic breast cancer cell model. The ultimate scientific goal of the Phase II contract studies is to apply the developed combinatorial RNAi screening technology to the large-scale discovery of all possible SL gene pairs in a set of 400 DDR targets from a panel of three cancer cell lines. As a practical outcome of the proposed studies, we will develop, validate and commercialize a novel open-resource SL DDR RNAi screening platform for the unbiased discovery and annotation of SL interactions in cancer cell models. To provide researchers with easy access to the combinatorial RNAi screening technology, Cellecta will offer custom bi-specific shRNA libraries and custom RNAi screening services in cancer cell models.